Cerebrospinal fluid from patients with melanoma brain metastasis shows global reconfiguration of cerebrospinal fluid immunokine profiles
On examining the overall levels of immunokines in our melanoma CSF samples, we observed a generalized suppression of multiple immunokines, including IL1α, IL1β, IL4, IL5, IL10, IL12, IL13, CCL5, CXCL9, CCL11, and CCL22, relative to the levels observed in controls, but not IL2, tumor necrosis factor-α, IFN-γ, CCL2, CCL23, CXCL1, and CXCL5 (Fig. 2b). In an effort to obtain a more global view of possible differences in the relative immunokine levels that may exist between melanoma patients and controls, we generated an unsupervised clustergram and heat map on the basis of the concentrations of the subset of immunokines found to vary between these two populations (Fig. 3a). The resulting analysis showed that melanoma patients were segregated away from the controls on the basis of their immunokine profiles. Generation of a hierarchical K-mean dendrogram from these data also showed a segregation of these two groups (Fig. 3b). These data suggest that the presence of tumor metastasis in the brain significantly alters host immunity within the central nervous system (CNS). Specifically, generalized suppression of IL1α, IL4, IL5, and CCL22 was detected in almost all melanoma CSF samples, suggesting the presence of global immunosuppression as part of a strategy aimed at evading host immunity against the melanoma metastasis. Furthermore, in a subset of patients, we observed the selective elevation of the three chemokines: CXCL10, CCL4, and CCL17, raising the possibility of selective chemokine activation for the purpose of oncogenesis.
Because tumors in the brain can have a disrupted blood–brain barrier, circulating chemokines in the blood could have leaked across the barrier, causing elevation of these chemokines in the CSF. Because we do not have concurrent serum samples from our cohort, we sought to answer this question by measuring the C-reactive protein (CRP), which is synthesized primarily in the liver and is present at a high concentration in the serum with the 95th percentile concentration at 9.50 μg/ml 21, but is generally excluded from the CSF. Compared with our reference nondisease serum sample with CRP measured to be 3.70 μg/ml, all of our melanoma CSF samples had CRP of less than 0.02 μg/ml (Fig. 2c), showing that intratumoral compromise of the blood–brain barrier was insufficient to explain the observed immunokine elevation in the CSF. In addition, immunostaining of the primary melanoma tumor from patient M3, who had a high level of CXCL10, showed staining in the tumor parenchyma (Fig. 2d), further suggesting the tumor origin of this chemokine.
It is possible that the differences observed between melanoma CSF samples and controls were because of previous therapies in the patient population. Dexamethasone use is an important temporalizing treatment for cerebral edema that is often associated with brain metastasis and the most likely therapeutic intervention that might account for our results. However, we found only nine of 22 patients were prescribed dexamethasone at the time of CSF collection, having a median daily dose of 4 (range 2–16) mg, and therefore dexamethasone is unlikely to be a contributing factor for the observed immunokine suppression. Furthermore, alkylating chemotherapies, such as dacarbazine, which is being used in conjunction with biologic agents (biochemotherapy), can potentially induce an immunosuppressive state in patients 13. Notably, serum levels of IL6, IL10, and IFN-γ in patients treated with dacarbazine-based chemotherapy combinations are significantly lower than those treated with dacarbazine-based biochemotherapy 22. However, in our cohort, only two patients received dacarbazine and one received thiotepa, whereas 12 patients did not receive any systemic treatment before CSF sampling. Therefore, alkylating chemotherapy is unlikely to cause the suppressed immunokine levels in our patient set.
Correlation between patient clusters and clinical outcome
We next asked whether different melanomas impose distinct immunokine signatures in the CSF. Indeed, unsupervised clustergram and heat map analyses suggested the presence of five separate clusters of patients with distinct immunokine profiles in our samples (Fig. 3a). As immunokines have been suggested to be important in driving tumor progression and metastasis 20, we therefore attempted to determine whether an association could be detected between any of these apparent clusters and patient outcome. We chose three epochs for our analysis: (a) time from diagnosis of melanoma to brain metastasis, (b) time from brain metastasis to death, and (c) overall survival. First, cluster 3 showed the shortest interval from melanoma diagnosis to brain metastasis, with a median time of 11.2 (range 0.0–306.1) months versus 31.2 (range 0.6–81.9) months for the rest of the melanoma cohort (P=0.2873, Table 3A). However, on closer inspection, this cluster contains the patient M21, who had a time interval of 306.1 months or 4.1 SD from the mean, who is clearly an outlier when compared with the other patients in our sample set (range 0.0–81.9 months). Upon exclusion of M21, cluster 3 indeed possesses a statistically significant shortened time from melanoma diagnosis to brain metastasis or 4.9 (range 0.0–17.5) months (P=0.0307). Notably, the remaining members in this recalculated cluster, M13, M19, and M22, all had elevated levels of CXCL10, CCL4, and CCL17, whereas IL1α, IL4, IL5, and CCL22 were markedly suppressed. Both CXCL10 and CCL4 are potent chemoattractants for CD8+ effector T cells 23–25, suggesting that these inflammatory proteins may play a role in promoting the formation of brain metastasis. Taken together, the CSF immunokine profile in these members of cluster 3 may support a propensity for the development of melanoma brain metastasis.
Second, we carried out an analysis to determine whether any of the clusters showed correlations with clinical outcome subsequent to the detection of brain metastasis. Cluster 4 showed a trend toward decreased time interval from brain metastasis to death with a median time of 4.1 (range 1.9–28.0) months versus 15.1 (range 2.6–73.3) months for the rest of the melanoma cohort (P=0.117, Table 3B). Interestingly, only CCL17 was elevated in all members of this cluster, whereas IL1β and IL6 were suppressed in addition to the commonly observed IL1α, IL4, IL5, and CCL22 immunokine suppression. Therefore, these patients’ apparent poor clinical outcome may arise from a more effectively tumor-subverted immune function relative to that in the rest of our melanoma cohort. Third, there was no detectable difference in the overall survival among the five patient clusters (Table 3C). This is likely because overall survival is influenced by the extent of the systemic malignancy rather than the number and size of the brain metastases or their treatment.
Correlation between patient clusters and previous biologics treatment
Because treatment with biologics and immune-checkpoint inhibitors can suppress or eradicate systemic melanoma 13–15, we next analyzed whether these interventions would alter patient outcomes or associate with any of the identified patients in our previous analysis. No difference was detected with respect to time from melanoma diagnosis to brain metastasis (P=0.6318), time from brain metastasis to death (P=0.3195), and overall survival (P=0.8538) on the basis of treatment with biologics such as high-dose IL2 and/or IFN-α. However, among patients who received biologics, those in cluster 1 appeared to show a marked shortening of the interval from diagnosis to brain metastasis, with a median time of 22.0 (range 0.6–38.9) months versus 34.7 (range 4.9–306.1) months for the rest of the melanoma cohort (P=0.1773). This trend may represent the selection pressure imposed onto the systemic melanomas that leaves some of the surviving clones with a higher propensity of metastasizing to the brain. Similarly, patients in cluster 4 who were treated with biologics show a trend toward shortened time from brain metastasis to death, with a median time of 7.8 (range 1.9–28.0) months versus 15.9 (range 5.2–39.0) months for the rest of the melanoma cohort (P=0.1275), suggesting that the brain metastases in this cluster are particularly aggressive after selection by biologics' treatment.
This is the first analysis of broad immunokine profiling in the CSF of patients with melanoma metastasis to the brain, a concept similar to immunoprofiling and establishing an immunoscore for systemic malignancies 26,27. This immunoscore may predict the efficacy of cancer treatments and pave the way for personalized immunotherapy 27. Indeed, our melanoma samples differed significantly from nondisease controls in cytokine and chemokine levels, including a marked suppression of IL1α, IL4, IL5, and CCL22 in almost all of our samples. It is important to note that although this constitutes a generalized suppression of immunokine levels as compared with control CSF, we also detected elevation of CXCL10, CCL4, and CCL17 in a large subset of our melanoma CSF. Immunostaining of the tumor origin of CXCL10, as well as our analysis of CSF versus serum CRP in control and melanoma CSF samples, also supports that these immunokine changes are specifically altered in the brain and do not emerge from the serum. Together, these data show a global response within the CNS to the presence of melanoma metastasis. There are potentially two explanations for this observation. First, this difference may reflect the altered activities of tumor-associated immune cells that impose immune suppression on the rest of the CNS through the secretion of soluble factors. This may result in suppression of resident immune cells, resulting in lowered levels of inflammatory cytokines observed in the current study 7. Such a general suppression has been shown previously for IL1β, IL4, and IL5 in melanoma-positive sentinel lymph nodes relative to melanoma-negative controls 28. Further analysis of this effect of melanoma metastasis to lymph node and brain could uncover more differences in immune modulation that may be specifically required for survival in these different sites. Second, it is possible that the downregulation of inflammatory cytokines that we observed could be a consequence of dexamethasone use or treatment by alkylating chemotherapies. However, our analysis showed that neither is likely to cause the observed immunosuppressive profile in the CSF. It is also possible that previous treatment with biologics may result in unpredicted responses in the immune system similar to those observed in our patient set. However, most patients, 16 out of 22, were treated with IL2 and/or IFN-α, whereas six were not, and there was no difference in the immunokine profiles between these two groups. Taken together, the immune suppression observed in our patients is likely to have been imposed by the metastases rather than arising as a result of previous therapies.
Another important observation derived from our data is that CXCL10 and IL8 are upregulated in the CNS of a majority of our melanoma patients. There is a striking, statistically significant 30-fold and 10-fold increase in CXCL10 and IL8, respectively, in melanoma CSF as compared with controls. We were able to detect CXCL10 immunohistochemical staining in the parenchyma of a primary melanoma, suggesting that the melanoma metastasis in the brain may also secrete this chemokine in an effort to recruit inflammatory effector CD8+ T cells into the tumor microenvironment for its own survival and proliferation. In addition, CXCL10 may be secreted by microglia and astrocytes. This is because CXCL10 upregulation has also been detected in Alzheimer’s dementia, which has an inflammatory component likely driven by microglia resulting in a protracted course of clinical deterioration 29,30. In experimental autoimmune encephalitis, the source of CXCL10 has been shown to originate from astrocytes within the brain, cerebellum, and spinal cord 31. Therefore, both tumor-derived and brain-derived CXCL10 may facilitate the survival and proliferation of melanoma brain metastasis. Furthermore, the IL8 chemokine is a potent mediator for angiogenesis 32–34. Melanoma tumor cells can also secrete IL8, but the level of expression may be regulated by the local tissue microenvironment 35. It is also secreted by activated microglia in the brain 36 and its level is elevated in the CSF of patients with acute and chronic inflammatory neurological disorders, including HIV-associated dementia 37 and opticospinal multiple sclerosis 38. Taken together, both tumor-derived and brain-derived IL8 may also facilitate the development of angiogenesis, which is critical to ensure the survival and proliferation of melanoma brain metastasis.
Despite the presence of generalized immunokine suppression in the melanoma CSF, there is enhanced expression of certain chemokines in specific patient clusters relative to nondisease controls. High levels of chemokines CCL4, CXCL10, and CCL17 (Group B in Fig. 3a) seem to aggregate together in the clustergram, and both CCL3 and IL8 chemokines also appear to cluster near these three chemokines. Notably, cluster 3 has the highest levels of CCL4, CXCL10, and CCL17, and it has the shortest time interval from melanoma diagnosis to the development of brain metastasis. CCL17, has been shown to be expressed by brain tissue and it is a potent chemokine for TH2-type CD4+CD25+ Treg cells because they have the corresponding CCR4 receptor 39,40. In patients with Vogt–Koyanagi–Harada disease, a rare autoimmune disease directed against tyrosinase and other melanocyte antigens that results in uveitis and neurological deficits, the CSF level of CCL17 was also significantly elevated when compared with control patients without the disease 41,42. Therefore, in this setting, overexpression of CCL17 may aid the recruitment of Treg cells that provide a counter-regulatory mechanism against the inflammatory reaction within the brain and eyes. It is also noteworthy that the serum level of CCL17 was lower in Vogt–Koyanagi–Harada patients than controls 41, suggesting that CCL17 is a chemokine specifically overexpressed in the brain. In melanoma patients, however, CCL17-mediated recruitment of Treg cells to the brain may attenuate antimelanoma protective immunity and enables tolerance to melanoma metastasis. Interestingly, certain melanoma cells also have the CCR4 receptor for the CCL17 ligand 40 and they may therefore co-opt the CCL17 chemokine axis for their own migration into the brain, suggesting a more complex role for this chemokine in promoting brain metastasis.
CCL3 and CCL4 are members of the IL8 chemokine superfamily 43 and both may therefore aid the survival and proliferation of melanoma brain metastasis. They are expressed in the brain during the acute phase of experimental autoimmune encephalitis, and neutralization of CCL3 by anti-CCL3 antibody limits the extent of brain damage in this model 44. In patients with ovarian carcinoma, elevated levels of CCL3 and CCL4 are associated with the presence of CD4+ T cells in the ascitic fluid, whereas melanoma patients had a predominance of CD8+ T cells in biopsy samples taken from the brain, lung, skin, and small bowel 45,46. These T cells most likely have a bias toward the TH1 response because CCL3 and CCL4 are known to activate antigen-presenting cells through the CCR5 receptor and during this process, IL12 is upregulated 47. However, within cluster 3, where CCL4 is elevated in all patients while CCL3 is high in some patients, only one member, M19, had elevated IL12 in the CSF, whereas the rest was average or low. The high CCL4 with or without elevated CCL3, together with low IL12, suggests that there may be yet unknown mechanisms of attenuating the TH1 response in patients with melanoma brain metastasis. Nevertheless, for patients in cluster 3, treatments that can drive down CCL3 and CCL4 may be useful therapeutic strategies. Furthermore, M21 is an outlier with the longest time interval within the entire patient set. In contrast to other members of the cluster, this patient’s CSF has a low level of CCL17 and a high level of IL1β. It is possible that the relatively lower level of CCL17 in M21 impairs the migration of melanoma cells into the brain, whereas elevated IL1β may be cytotoxic to the ones that arrived there by means other than the CCL17 chemokine axis and others that survived there because of impaired TH1 adaptive immunity 40,48. Therefore, treatment that can lower CCL17 levels may prevent the development of melanoma brain metastasis.
Members within cluster 4 have suppressed IL1β and IL6 cytokines in addition to the generalized IL1α, IL4, IL5, and CCL22 immunokine suppression. Furthermore, only CCL17 chemokine was increased in this cluster. Patients within this cluster have poor survival once brain metastasis is established, irrespective of previous biologics treatment. We speculate that the severe immunokine suppression in the CSF represents a similar state of immunosuppression within the brain that provides a favorable environment for melanoma brain metastases to grow and proliferate. The extent of suppression is somewhat surprising and may arise from the immunologically sequestered nature of the CNS where smaller amounts of tumor-derived immunosuppressive factors can achieve a global effect. Finally, cluster 1 has a trend for shortened time from melanoma diagnosis to brain metastasis and this only occurred in those who received biologics treatment. It is possible that biologics treatment places selection pressure on the systemic melanoma and that the surviving clones have a high propensity of metastasizing to the brain. However, both observations need to be validated in a larger population of melanoma cohort.
A major limitation of our observations is the small sample size, which is based on 22 individual CSF samples. This likely contributes toward the lack of statistical significance in the prognostic significance of our patient clusters on the basis of a set of immunokines. However, the robust segregation of melanoma CSF from controls, as seen in both the clustergram/heat map and the K-mean dendrogram analyses, strongly suggests that melanoma metastasis to the brain causes global changes in the immunokine milieu within the CNS that can be detected in the CSF. Notably, there is generalized immunokine suppression, whereas specific CXCL10 and IL8 chemokine levels are increased. Therefore, these findings provide the necessary foundation for the identification of immunokines and their relative levels of expression in the CSF, as well as their potential utility as diagnostic biomarkers for melanoma brain metastasis. Furthermore, as more is known about the immunological phenomena associated with tumors in general and within the CNS, new treatments can be developed to interrupt these tumor-associated manipulations of the immune system.
The authors acknowledge the support from A Reason to Ride research fund. K.D.S. was supported by R56AI085131 from the National Institute of Allergy and Infectious Diseases (NIAID).
Conflicts of interest
There are no conflicts of interest.
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Keywords:© 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins
brain metastasis; cerebrospinal fluid; chemokine; cytokine; melanoma